Transgenic Mitomice Models for Treatment of Blinding Diseases

描述(申请人提供)：虽然突变的线粒体(Mt)DNA与Leber遗传性视神经病变(LHON)的失明密切相关，但具有突变的mtDNA复合体I亚单位的真实动物模型尚未开发出来，该模型将能够探索视神经病变的发病机制和测试潜在的治疗途径。由于在将DNA输送到线粒体方面存在障碍，因此不可能建立由线粒体突变引起的人类疾病的动物模型。我们通过在腺相关病毒(AAV)上附加一个线粒体靶向序列(MTS)到病毒蛋白衣壳上来绕过这一障碍，将突变的人ND4基因(负责所有LHON病例的一半)引导到小鼠线粒体中。在人类线粒体启动子的驱动下，突变的人ND4在小鼠线粒体中的表达导致视力丧失，视网膜及其组成视神经的轴突中的神经节细胞进行性死亡，从而概括了人类LHON疾病的特征。我们从事这个项目的动机是为导致失明的无法治愈的线粒体疾病开发基因疗法。为此，我们必须(1)开发一种可行的治疗方法，即MTS AAV，将正常基因输送到受影响的线粒体；(2)创建具有突变的复杂I亚单位基因的动物模型，以概括人类疾病，在其中测试潜在的治疗方法，并在类似人类疾病的阶段测试视神经疾病的发病机制。为了获得转基因小鼠，我们将含有突变的人ND4等位基因的MTS AAV通过显微注射到小鼠的胚胎干细胞中，产生了出生一年后视网膜神经节细胞(RGC)进行性死亡和视神经病变的后代，其模式视网膜电信号(PERG)幅度稳定地下降到噪声水平。为了监测线粒体基因在活体动物中的表达，我们在含有突变型人ND4的MTS AAV结构中添加了线粒体编码的红色荧光蛋白(MCherry)，这种突变可以通过激光扫描眼底镜(LSO)在活体后代及其迄今的三代后代中观察到。我们的具体目标从了解突变的NADH脱氢酶亚单位ND4在小鼠中的后果发展到开发额外的转基因株，表达另外两个突变的人类复合体I亚单位(ND1和ND6)，这些突变的人类复合体I亚单位负责LHON。

DESCRIPTION (provided by applicant): While mutated mitochondrial (mt)DNA is firmly linked to the blindness of Leber's hereditary optic neuropathy (LHON), a bona fide animal model with mutated mtDNA complex I subunits that would enable probing the pathogenesis of the optic neuropathy and testing of potential avenues for therapy has yet to be developed. It had not been possible to produce animal models of human diseases caused by mitochondrial mutations due to the barrier in delivering DNA into mitochondria. We circumvented this barrier by appending to the adenoassociated virus (AAV) a mitochondrial targeting sequence (MTS) to the viral protein capsid to direct the mutant human ND4 gene (responsible for half of all LHON cases) into murine mitochondria. When driven by a human mitochondrial promoter, expression of mutant human ND4 in murine mitochondria induced visual loss with a progressive demise of ganglion cells in the retina and their axons comprising the optic nerve, thus recapitulating the hallmarks of the human LHON disorder. Our motivation in pursuing this project is to develop gene therapy for untreatable mitochondrial diseases that lead to blindness. For this, we have to (1) develop a plausible therapy, an MTS AAV to deliver normal genes to affected mitochondria and (2) create animal models with mutated complex I subunit genes that recapitulate the human disorder in which to test potential treatments and the pathogenesis of optic neuropathy at stages that resemble the human disease. To generate a transgenic mouse we delivered the MTS AAV containing the mutant human ND4 allele to embryonic stem cells by microinjection into the blastocyst of the mouse, generating offspring with progressive retinal ganglion cell (RGC) demise and optic neuropathy with pattern electroretinogram (PERG) amplitudes declining steadily to noise levels one year after birth. To monitor mitochondrial gene expression in live animals we added a mitochondrial encoded red fluorescent protein (mCherry) to the MTS AAV construct containing mutant human ND4 that could be visualized by laser scanning ophthalmoscopy (LSO) in live offspring and so far in three generations of their progeny. Our Specific Aims logically progress from understanding the consequences of mutant NADH dehydrogenase subunit ND4 in mice to developing additional transgenic lines expressing the two other mutant human complex I subunits (ND1 and ND6) responsible for LHON.